Historically, wine fermentations were permitted to run their course, using only the diverse wild yeast strains that were indigenous to the fruit's surface. That practice has for the most part been replaced in commercial wine fermentation processes, that today rely instead upon the controlled production and use of pure or selectively mixed yeast strains. The pitching of beer in modern brewery operations is also carried out using carefully produced, high purity, pitching masses.
It has been proposed that commercial fermentation practices might next evolve to the point where immobilized cells could be routinely used. Some encapsulation methods employ organic solvents or other reagents that are strictly incompatible with many potential biological encapsulants.
The use of various gel-forming proteins (collagen and gelatine) and polysaccharides has resulted in the development of milder, more biocompatible immobilization techniques. Note, however, that in the case of agar and carrageenan, for example, such procedures can involve heating the polymer to a temperature where it is in a liquid state (e.g. up to 60 degrees C.), at which point the immobilizant is added, and whereafter the resulting mixture is solidified by cooling. The exposure of a biological immobilizant to the requisite elevated temperatures may be undesirable.
Gentler still is the simple gelation immobilization technique that was developed for and is used primarily with alginate. Generally, this technique involves the drop-wise addition of the ionic polysaccharide/immobilizant solution, through a syringe needle, into a solution of a divalent cation. The divalent ions cross-link various charged species on the polysaccharide molecule, with the result that insoluble gel beads are formed. Where alginate is selected as the ionic polysaccharide, as is typically the case, divalent calcium ions are employed as crosslinkers. This "syringe extrusion" process has the advantage of producing beads having a narrow, unimodal size distribution.
While it is known that yeast cells can be entrapped in calcium alginate and that the resulting immobilized yeast can be used in expediting fermentation processes, and while there are numerous references that are concerned with this advance, there are very few commercial applications.
There are a number of reasons for this. Alginate beads are very frangible, and their usefulness in commercial scale operations is therefore easily compromised. For example, being as they are so very soft, the beads are easily compressed. Commercial scale operations require large scale fermentation columns, such that in down-flow fermentations the beads are difficult to keep, from compacting, while in typical up-flow fermentations the beads are very prone to ablative wear and channelling.
Moreover, in the presence of, for example, citrates, lactates, and phosphates, alginate beads become prone to Ca.sup.++ ion loss through chelation.
In commercial scale operations, the problem with alginate entrapment of cells also arises out of the manner in which the particulate gels are formed. The processes must be carried out at the production site where a yeast slurry and a solution of sodium alginate are mixed together. When this mixture is subsequently fed, dropwise, into a calcium salt solution, the sodium alginate is displaced as a calcium alginate salt gel, which over the course of the gelling process, occludes the suspended yeast cells within the gelling salt particles. Beads of uniform size and quality can be produced using this technique in conjunction with the above mentioned syringe extrusion process. Bead size, however, is limited by the syringe needle bore size and viscosity of the solution. As a result, beads of less than 3 mm, and especially beads of less than 1mm can be difficult to produce. At the same time, however, smaller diameter beads are needed to better facilitate both internal and external mass transfer, enhancing fermentation performance and minimizing bead rupture due to gas formation and accumulation. Production of small bead sizes has been attempted previously by modifying the syringe extrusion process, through the use of air jets impinging on the needle, electrostatic pulses, or vibrating needles.
The problem with unmodified alginate syringe extrusion bead production techniques that do not utilize one or another of these bead size reduction variations, is that economical production of immobilized beads is limited to large (3 mm to 5 mm) beads. The size of these large beads imposes diffusion limits on the transfer of substrate and product to and from the entrapped yeast cells. For example, in some cases the diffusion problems allow anaerobic fermentations to take place internally of the bead, notwithstanding the fact that to all outward appearances, aerobic fermentation is proceeding normally at or near the bead surface.
For large bead production (e.g. 3 mm) these can be formed by a single droplet generating technique, for which production rates may reach 24 l/h per syringe needle. Multiple needle extruders can even support small-scale industrial production rates.
On the other hand, while the variations on the syringe extrusion technique mentioned above can be employed for facilitating smaller alginate bead production, they too are-fraught with economic penalties. These include needle like extruders of one of two typical designs. The first such involves producing small drops of sodium alginate/yeast slurry, by passing the material through the needle, and with a vibratory action, shaking off a smaller drop than would otherwise form in the absence of the vibration. The second approach uses a coaxial flow needle, in which the solution of sodium alginate/yeast is passed through the centre of the needle, and as droplets form at the end of the needle, a coaxial flow of air pulls a small droplet away from the tip. These approaches can be used to keep alginate bead sizes as low as possible (with standard deviations of about 20%). However, the number of needles needed to maintain the flow rate is inversely proportional to the bead volume. Reducing the bead size to 500 micrometers or 100 micrometers requires the use of several hundreds or even hundreds of thousands of needles operating concurrently: a complex, expensive, and generally awkward solution. On the other hand, however, even these methodologies provide only very limited rates of bead production throughput, and are accordingly very difficult and expensive to scale up sufficiently to supply commercial fermentation processes.
Attempts have also been made to form more durable carrageenan beads utilizing the above "dropwise" methods. In the practice of this process, a type of carrageenan is employed, from which indigenous potassium ions have, been removed, (as for example, by way of ion exchange treatment). Droplets of this polymer are then extruded into a potassium ion-containing solution, to effect the gellation of the carrageenan beads. This method, however, results in the gel forming from the surface of the droplet, so that a gel membrane sets up around the droplet at first, and it is only as the salts diffuse inwardly through the ever thickening membrane, that the interior of the droplet gels up. As a consequence, the resulting gel structure of the bead that is formed in this manner is not as homogeneous as might be desired.
The production of beads by polymerizing emulsions in which droplets, comprising an aqueous suspension of a prepolymer and cell mixture, forms the discontinuous phase that is dispersed, using propeller type mixers to form the emulsion of droplets in a continuous oil phase, has also been attempted.
U.S. Pat. No. 5,079,011 teaches away from the use of alginate, citing the various short comings that are set out hereinabove. This U.S. Patent teaches instead, that a fibrillar matrix can be employed to immobilize the yeast cells, and cites the non-compressible nature of, for example, DEAE cellulose as demonstrative of its superiority to the weak physical structure that is associated with the aforementioned alginate. Note, however, that the immobilization of yeast cells in accordance with the materials promoted by the subject US patent, is limited to surface immobilization. This means that the amount of immobilized yeast is a function of bead surface area, and not of bead volume--and accordingly, that the reactor volume must be correspondingly larger (or the fermentation correspondingly slower). DEAE cellulose, moreover, is best suited for packed bed fermentations--it is not well suited to fluidized bed applications because the immobilized biomass is prone to detachment at the higher shear rates. Accordingly, DEAE cellulose surface immobilized yeast cells are not suited to primary fermentation in the production of beer, where the enhanced external mass transfer rates associated with fluidized bed reactors is desirable, if not necessary to commercial scale operations.
This latter problem also arises in prior art attempts to use carrageenan as a carrier. In Example 1 of PCT/US88/03980, the use of carrageenan beads is disclosed. Porous carrageenan beads having average diameters between 4.0 to 4.5 mm were obtained from Fisher Scientific and the yeast cells were then immobilized onto the bead surface, by way of incubation at 37 degrees C., for two hours in a shake flask.
An attempt to produce a carrageenan bead with yeast entrapped therein, using alginate bead production equipment, has not proven to be satisfactory. The alginate-bead producing equipment normally employed a rotatory disc atomizer to spray sodium alginate-containing droplets into a centrifugal bowl containing a suitable calcium salt. The alginate beads form in the bowl and the centrifugal rotation of the bowl carries the formed beads over the lip of the bowl, and into a collection vessel (typically the fermenter itself). When the equipment was utilized in the attempt to produce carrageenan beads, it was found that the viscosity of the carrageenan solutions that were sufficiently concentrated to avoid the mechanical problems associated with alginate beads, was much higher than the viscosity of the alginate solutions, (i.e., 2000 centipoise for the carrageenan versus 200 centipoise for alginate solutions). Accordingly, the alginate bead making equipment did not perform at all satisfactorily, either with respect to the bead quality or the size distribution of the resulting carrageenan beads.
Accordingly, there remains a need in the art for an economically practicable process that will enable physically durable carrageelnan beads of relatively small sizes to be produced with yeast cells entrapped therein.